Recently there has been progress with respect to obtaining information on the relation between enzymes and diseases. Accordingly, there is a general need to assay the activity of enzymes in vitro in order to diagnose diseases. ChE is one enzyme typically assayed in vitro. Its activity is closely related to liver function. An assay of its activity is generally carried out to diagnose liver disease.
In clinical laboratories or the like, ChE is at present assayed by, for example, the following processes:
(1) a process of using acetylcholine as a substrate and measuring the change in pH;
(2) a process of using thiocholine ester as a substrate and measuring the amount of released SH group; and
(3) a process of using benzoylcholine or the like as a substrate and measuring the amount of released choline as H.sub.2 O.sub.2 using a choline oxidase.
Process (1) is a standard process which has been employed in laboratories for a long time. However, its accuracy is poor because it is difficult to maintain the optimum pH and any pH-change does not necessarily correspond to the degree of change in color of an indicator. Accordingly, processes (2) and (3) are taking the place of process (1). However, process (2) still has a problem of error due to the coexistent SH compound, and process (3) has problems in connection with determination of H.sub.2 O.sub.2 and a problem because phenol in color formation causes interference with the assay system. Thus, no satisfactory processes have been developed. These processes are also inconvenient because even though activities assayed by these processes are presented in terms of "international units", the normal level range of ChE varies greatly depending upon the process employed, resulting in confusion in laboratories. Accordingly, there has been a need for the standardization of the assay of ChE and there has been a tendency to examine process (1). Under the abovedescribed situation, a more accurate process based on process (1) could be the most satisfactory process. However, no such processes are presently known.
One known process involves using acetlycholine as a substrate. Two enzymes of an acetate kinase and a pyruvate kinase act on acetate released from the substrate by the action of ChE to thereby produce pyruvate. The pyruvate is then reacted with a chromogen to produce a dye which is measured by colorimetry (T. Takatori, Japan. J. Clin. Chem., Vol. 4, No. 2, 1975, pp. 186-189). However, this process is not desirable because non-thermostable acetate kinase yielded by bacteria growing at ordinary temperature (Escherichia coli) are used. Accordingly, the usable concentration range of the acetate kinase is extremely narrow. Therefore, the values obtained fluctuate, greatly making reproducibility poor. The accuracy of the process is not good, making the process impractical.